The objective of this application is to determine the mechanism by which estrogen withdrawal, seen in aging women, increases LXR alpha (a gene necessary for regulating cholesterol homeostasis) transcript levels. My central hypothesis is that the estrogen-bound estrogen receptor (ER) represses AP-1 dependent activation of LXR alpha transcription. This hypothesis is based on our recent findings that a decrease in estrogen significantly increases the level of LXR alpha mRNA/protein in primary macrophages and in the monocytic-macrophage cell line THP-1 in the absence of nascent protein synthesis. In addition, this hypothesis incorporates data from other labs that estrogen, through the ER (predominantly ER beta) can repress transcription at AP-1 consensus sites in macrophage cell lines and that the LXR alpha promoter contains multiple AP-1 binding sites but not an estrogen response element. To test our central hypothesis aim one will determine ER's role in regulating the LXR alpha estrogenic response by quantitating transcription after treating macrophages with antiestrogen (ICI 182,780) and HPTE (ER alpha agonist; ER beta antagonist) in the presence and absence of lX10-8 M 17-beta-estradiol. Aim two will determine transcription factors impacted by estrogen that act directly in regulating LXR alpha gene transcription in macrophages. Luciferase activity driven by an intact or mutated (e.g., AP-1 binding sites) LXR alpha promoter will be measured in transiently transfected macrophages before and after estrogen treatment. Once specific promoter sequences are identified that transduce an estrogen signal the activity and binding of specific proteins (e.g., Jun and Fos family members) to small labeled promoter sequences (about 20 bp) will be determined by western analysis and electrophoretic mobility shift assays (EMSA), requiring nuclear extracts of THP-1, treated with and without estrogen. The expected result will identify AP-1 binding site(s) and identify the individual proteins that make up the AP-1 complex that binds to the AP-1 promoter element required for increased LXR alpha transcription after estrogen depletion in macrophages.